Genetic manipulation of Schistosoma mansoni: Transgenesis and RNA interference
Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. It is considered the second most important parasitic disease of humans, after malaria. The WHO Schistosoma Genome Project was initiated in 1994 with the declared goal to identify target genes in order to be able to address fundamental questions on the biology and physiology of the parasites, and to guide the rational development of drugs, vaccines and other intervention strategies. Nonetheless, despite this abundance of sequence data, true functional analysis of potential target genes will only be possible when a reliable method for reverse genetics in schistosomes becomes available. At the outset of this project, we hypothesized that the genomic structure of the cathepsin D-like protease involved in digestion of hemoglobin with in the gut of Schistosome mansoni is of similar complexity to aspartic proteases of some other eukaryotes and that the upstream regulatory sequence of the schistosome cathepsin D gene is capable of inducing transient reporter gene expression in mammalian cells. We also hypothesized that the aspartic protease cathepsin D is an important enzyme for the normal growth of these parasites in vitro and that it is an essential gene needed in the schistosomula stage to grow into adulthood in mice. Finally we hypothesized the transposable element piggyBac can mediate genomic integration and expression of transgenes in schistosomes Cathepsin D-like protease, the apical enzyme of the hemoglobin proteolysis cascade in schistosomes was investigated. First issue addressed was the gene structure of cathepsin D of S. mansoni. The locus of this gene was located within a bacterial artificial chromosome (BAC) constructed from S. mansoni genomic DNA, and its genomic structure characterized. The exon/intron arrangement of the schistosome cathepsin D gene was compared and contrasted with homologues from model species from informative eukaryote taxa including yeasts, apicomplexans, nematodes and vertebrates. Secondly, transcriptional silencing of the cathepsin D gene was analyzed in detail, including short term and long term exposure to dsRNA. Parasite age and susceptibility to the dsRNA were addressed as were RNAi effects in vitro versus in vivo. Finally, to determine whether transposon could mediate somatic transgenesis in S. mansoni, we examined whether the piggyBac transposon could deliver reporter transgenes into the genome of S. mansoni parasites. A piggyBac donor plasmid modified to encode firefly luciferase under control of schistosome gene promoters is introduced along with 7-methylguanosine capped RNAs encoding piggyBac transposase into cultured schistosomula by square wave electroporation. Luciferase activity in transformed schistosomules was analyzed, and piggyBac integration sites within schistosome chromosomes were characterized These findings provided in this dissertation are the first direct evidence of somatic transgenesis of schistosomes, or indeed of any parasitic helminth